Mosquito-borne members of the Flavivirus family include four serotypes of dengue virus (DENV1-4) and West Nile Virus (WNV) and are classified as re-emerging pathogens due to the frequency and severity of recent epidemics. There are an estimated 100 million DENV infections reported each year resulting in nearly ~25,000 deaths. More than 2.5 billion people are at risk of infection by DENV. Symptoms of dengue fever include fever, rash, and arthralgia. In more severe cases, viral infections can develop into life-threatening dengue hemorrhagic fever or dengue shock syndrome. Dengue shock syndrome occurs when leakage and/or bleeding is sufficient to induce shock, which often leads to death, especially in children, due to the lack of adequate supportive care. WNV infections can result in meningitis, encephalitis, or paralysis leading to ~10% mortality among hospitalized patients. Year 2012 witnessed a sudden surge in the number of WNV infections, comprising >5,000 cases in 48 states with ~228 deaths. Despite the high morbidity and mortality resulting from flavivirus infections in a subset of patients, there is currently no effective chemotherapeutic treatment for infections for any of the flaviviruses. The combined global socioeconomic impact of flavivirus pathogens merits an urgent need for new and effective antiviral therapeutics. The Flavivirus RNA genome encodes for three structural proteins (C, prM and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). The NS3 protein is a multifunctional enzyme that contains protease, helicase and RNA triphosphatase activities. The N-terminal region of NS3 has a serine protease domain, which in the presence of the NS2B cofactor (NS2B, is an active serine protease. This protease (NS2B-NS3pro) plays an essential role in the cleavage of the viral precursor polyprotein and disruption of this function is lethal t viral replication. Therefore, blocking viral replication by inhibiting the quintessential viral protease could lead to the development of effective broad-spectrum antiviral therapeutics and is the focus of our research proposal. The innovation of our proposal is the discovery of two lead inhibitors, which have broad-spectrum (DENV1-4 and WNV) inhibitory activity and contain a catechol moiety, which is also a substructure present in 17 FDA approved drugs. In addition, the modeling data suggest the compounds bind to the enzyme substrate binding pocket and thus act as competitive inhibitors. Therefore, these properties of our leads support the feasibility for development into effective broad-spectrum antiviral therapeutics. The specific aims for Phase I of the proposal are: 1. Design and synthesize broad-acting inhibitors of DENV1-4 and WNV NS3pro; Milestone: Compounds with IC50d100nM against DENV1-4 and WNV protease will be advanced into cell-based assays. 2. Evaluate the therapeutic indices of optimized lead compounds using in vitro cell-based replicon, infectivity (EC50), and cytotoxicity (CC50) assays; Milestone: Identify 15-20 lead compounds with EC50d200nM and CC50e200uM representing the catechol and non-catechol series. 3. Conduct in vitro ADME-based lead optimization of compounds with broad-spectrum in vitro activity and acceptable therapeutic indices; Milestone: Identify 5-10 lead compounds exhibiting high bioavailability, weak inhibitor of CYPP450s, optimum stability and are not hERG channel blockers for evaluation in in vivo animal models for acute toxicity, efficacy and pharmacokinetics (Phase II application).